Method for testing for polymorphonuclear leukocytes



United States Patent ()fiice 3,102,418 Patented Sept. 3, 1963 Oscar W. Schalm andDaniel 0. Noorlander, Davis, Califi,

assignors to The Regents of the University of California Berkeley, Calif.

No Drawing, Original application June 18, 1956, Ser.

No, 591,777, new Patent No. 2,998,392, dated Aug. 29,

1961. Divided and this application Sept. 12, 1960,

Ser. No. 56,630 1 3 Claims. (Cl. 73-53) This invention relates to and in general has for its object the provision of a method for testing the presence of mastitis, and this application is a division of our application Serial No. 591,777, filed June 18, 1956, for Method and Solution for Testing for Polymorphonuclear Leukocytes, now Patent No. 2,998,392 Mastitis is an inflammation of the mammary glands caused by the infection thereof with various bacteria capable of causing disease and which currently is very common in machine milked commercial dairy herds. The occurrence of pus in milk indicates the presence of mastitis in the mammary glands.

Presently, there are available various methods for testing for mastitis in the field, although for one reason or another none of them is entirely satisfactory. One such method, for example, involves the use of so-called strip cups provided 'with IOO-rhesh screen false bottoms and through which milk is drawn for the detection of visible particles. However, to be effective as a means for detecting chronically irritated mammar/ glands, this test must be used daily, for in chronic mastitis the milk is not visibly abnormal at all times. The milk from a given gland may show positive on one strip cup examination at one milking but not on the next.

Furthermore, a single use of the strip cup method seldom the chronically detects more than to 20 pe'icent of inflamed glands.

Another field test sometimes resorted to involves the use of blotting paper impregnated with bromthymolblue for detecting abnormal acidity or alkalinity in the milk This test at best does not detect more than 30 to 40 percent of the chronically inflamed glands, and when the inflammation has become of suflicient magnitude to alter the pH of the milk or its physical appearance, usually I considerable damage to the mammary tissues has already occurred.

More specifically, the object of this invention is the,

any desired quarter of a cow with about an equal quantity of an aqueous solution of an anionic surface active agent containing a trace of a contrasting dye and observing :the mixture for the occurrence of thickening leading to an actual gel formation in very positive samples.

The following reagents are illustrative of the anionic. surface active agent suitable for this purpose: a sodium or potassium salt of a fatty acid, a sodium or potassium alkyl aryl sulfonate, a sodium or potassium alkyl sulfonate, a'sodium or potassium alkyl aryl sulfate, or a sodium or potassium alkyl sulfate. More specifically, we have found that an aqueous solution (distilled water) of from 2 /2 percent to 3 percent of an anionic surface active agent containing a dye such as bromcresol purple or bromthymol blue in a final concentration of active, agent in the order of 5 percent does not interfere with the test, and as little as 1.5 percent of the agent gives a reaction with positive milk, although of a somewhat lesser intensity.

It should also be here observed that many soaps and synthetic detergents are primarily anionic surface active agents and therefore can be efliecti-vely used as a mastitis test solution along with a small amount of a suitable dye for rendering more visible the reaction thereof with milk being tested and thus making it possible to grade such reaction. This type of solution is suitable not only for testing for mastitis but also for determining the alkalinity of the milk.

Converiientlyfour method can be carried out by the use of four shallow plastic cups having a common white opaque base and upstanding therefrom, the cups being arranged in a square pattern so that milk from each quarter of a cow can be selectively delivered to each of about euflicient to cover its bottom, the quantity of testto and having a substantially neutral pH serves are not critical in that an excess of the anionic surface 0 that the proportions or concentrations above set forth 'ing solution to be added to each cup can be readily gauged with a suflicient degree of accuracy for the purposes of the (test Without requiring the use; of droppers or other metering equipment. 7

If an abnormal count of polymorphonuclear leukocyte cells is present in the milk it will thicken almost immediately upon agitation of the mixture; the degree of thickening parallels the PMN leukocyte count. A slight thickening, which tends to disappear as agitation of the sample is continued, is called a TRACE reaction; a thickening without gel formation is a one plus reaction; when the mixture thickens and tends to gel and exhibits a tendency to be drawn toward the center of the cup as it is moved in a circular fashion, the reaction is called two plus; and, finally, milk that forms a gel or very viscous mass which tends to adhere to the bottom of the cup and to have a convex or bulging upper surface, is classified as three plus reaction.

Although the bases of the testing cups have been referred to as opaque, some light can pass through them to partially illuminate the film of milk contained therein,

this being of importance when milking is done in a rela- I tively dark barn.

Asrabove indicated, the dye used should be of a color contrasting with the color of the milk and with the base of the cups. The dye in the test solution aids in ascertaining when the proper amount of test solution has been added to the milk, for as soon as the white color of the milk has disappeared, sufficient test solution has been added. Also, it is to be noted that the dye used is a pH indicator, and that since the test solution has a neutral pH, upon the addition thereof to the milk, the color of the solution will change if the milk be either on the acid or the alkaline side.

Although the use of the cups above described is convenient in making our test for the reason that it is easy to bring them to a uniform milk content, to identify them relative to the four quarters of a cow, and to readily observe what takesplace when the test solution is added thereto, test tubes or other receptacles may of course be used.

To gain a better understanding of the significance of the positive reaction of milk to our solution, consideration should be given to the following information.

types of epithelial origin. Any significant occurrence of true polymorphonuclear leukocytes is an indication of an abnormal condition.

work in Sacramento County, California; and, colostrum is material collected from the udder on the day of calv ing;

With each type of milk, the mean cell level at which single milking; mixed milk with the preservative boric acid is milkthat is 12 to 36 hours old and that has been collected by a cow sampler ,in Dairy Herd Improvement V The number of cells in milk is subject to wide variation .a one plus reaction begins is diiferent. suggests under physiologic conditions. In colostrum, cell fragthat PMN leukocytes are not solely responsible for the ments or cells laden with fat, the so-called colostrum correaction but that other products of inflammation are puscles are of common occurrence, Whereas in the dryingprobably also involved. Cell counts are highest in ioreofi secretions a high cell count due to sloughing of epi milk, strippings and colostrum, whereas when mixed thelial cells is to be anticipated. udder milk is used dilution occurs so that the counts are The cells in milk are expressed in number/cc. of milk. somewhat lower at the point where a definite reaction Among 690 normal milks from individual mammary develops. However, mean total cells per cc." of milk quarters, collected from the second week through the in every instance, even'in the mixed udder milk, exceed seventh month of lactation, the following percentage dis- 500,000 cells which was the ripper limit of the cell count tribution in total cell counts was recorded. '15 in 96.5 percent of 690 known normal milks. Furthermore, these data reveal that the ratio of PMN leuko- Percentage cy'tes to mononuclear cells in normal milks is about 1 to Number of cells/cumin fifig 2, whereas with milks positive to our mastitis test the ratio is approximately 1 to l with one plus reaction, 1 to (H00 7M2 0.6 with milks giving a 2 plus reaction, and l to 0.4 100, ob -250,000 13.76}00. 51 with milks giving a three plus reaction. This means 538035503 3 3 31g? that our mastitis test reflects the occurrence of PMN over 1, 000, 000"... 1.15 leukocytes in above normal numbers in milk, and there- 99 97 fore it is a rapid, inexpensive procedure for the detection of abnormal milk. 1 I

Differential cell counts on normal milk showed that counting 9 {cells l 1s leibonous l 81.6 Percent of the milks had less than 25 percent of the fore lmpractlcfnd for mutme l 031s WW? a cells of polymonphonuclear (PMN) type and in '93 Peri g g t s ggi g g 2; g 130st 2 5 3 cent of the milks, the PMN type cell contributed less than s 9 r e e c 10111 d eFexls 0 percent of the total cells. Of all normal milks studied, 00101210911 F mammary g an or eXamP e! 96.5 percent had total cell counts not exceeding 500,000 L1t 1e (B 1, A companson of the bacll per c( f ilk ttenological and physiological reactions of normal and Cell counts have been made on several hundred milks mastitic milk from young COWS, Cornell Vetermarian, that have been tested using our test solution. Data thus 35 VOL 1938, par d =pH, chl and 1001(0- far obtained are as follows: cyte number of normal milk with milk from quarters Type of milk negative trace one plus two plus three plus 5 PMN 10, 000 200,000 000,000 I 2,130,000 3,730,000 Fore'milk (200 samples) MONO 38,000 400,000 000,000 1,370,000 1, 000, 000 SSH herd ratio 1 to 3.8 '1 to 2 1 to 1.1- 1 to 0.04 1 to 0.47 JPMN 48, 000 110,000 330,000 1,320,000 0, 000, 000 Mixed udder milk (113 samples) lMONO--- 91,000 200,000 300,000 050,000 2,500,000 so herd ratio 1 to 1.9 1 to 1.8 1 to 1.1 1 to 0.5 1 to 0.4

86,000 180,000 430, 000 1,550,000 Mixed udder milk with preservative (borlc'acid) 97 samples, 200,000 240,000 420,000 750,000 1 to 2.3 1 to 1.3 1 to 0.87 1 to 0.48 86,000. 05,000 550,000 1, 920, 000 3,000,000 .Mixed udder milk (53 samples)...

130,000 170, 000 500,000 1,240,000 1,410,000 UCF herd 1 to 1.5 1 to 2.6 1 to .9 1 to .64 1 to .4 PMN 200,000 I 800,000 2,000,000 0, 100, 000 Colostrum (25 samples) 1 330, 000 l 500,000 1,170,000 2,500,000 SSH herd 1 to 1,40 1 to 0.02 1 to 0.53 1 to 0.4

' 29 1 stnppmgs Sump es) 000, 000 750,000 1,520,000 ssH herd 1 to 1.7 1000.83 1 to 0.29

f 1 PMN type eell=1.

1 N0 samples.

.Ihe data presented above are in terms of the mean known to be infected with pathogenic streptococci. The cell count of PMN leukocytes (plus cells) and monofollowmg results were reported: 4 1 nuclear cells (essentially epithelial of cells of mammary v gland origin) for each of the live classifications of restrepto, actions occurring with milk when milk and our Mastitis giffi 4 Test Solution are mixed in equal parts. Fore-milk is the percent milk, 'first streams of milk taken from the gland before a regu- 7 m lar mllkmg period; strlppingsrepresent m1lk re rna1mn g 1n pH of fowmilk o-ver M the slander the Completwn of regular g; rmxed Chlorides over 0.15 grams percent- 0.5 3. udder milk is a sample taken from the bucket and reprei gfllllsfififi itu "37.0 sents the mixed milk from the entire udder obta ned at a over 500,000 cel1s/cc.m1lk -1 4.0 90.

Murphy (The general effect of staphylococcal infection on the biochemical composition of strict toremilk,

Cornell Veterinarian, volume 33, 1943, 52), compared the leukocyte, chloride content, and pl -I of fore-milk coming trom glands which he classified as uninfected, Staphylococcus-infected and Streptococcus-infectedz The data assembled by Murphy clearly show that streptococci more commonly causes alterations in the milk than micrococci. This has been the experience of all observers who have compared the two types of infections and this difference was described as early as 1918 by Jones (Studies in Bovine Mastitis, I, Non-hemolytic streptococci inflammation of the udder, Journal Experimental Medicine, volume 28, 1918, 149).

Adler and Migaki (-Cell types in milk cows in a chronic staphylococcal mastitis herd, Veterinary Medicine, volume 46, 1951, 89), in a study of cell types in milk of cows in a chronic staphylococcal mastitis herd concluded: In Staphylococcus pyogenes infection there is a marked increase in P-MN leukocytes; this increase is apparent even though the condition has not yet advanced to a clinical state and is a more sensitive indicator of the mastitic condition than either a total cell or bacterical count.

These literature references which are representative of the many reports that have appeared clearly indicate that when bacterial infections of the udder reach the point where irritation or inflammation is produced, the cell count and especially the PMN leukocyte number rises. Any factor that Will produce irritationin the bovine mammary gland, whether bacterial or physical (forceful striking of the udder or allowing milking machine vacuum to injure the udder) will elicit an inflammatory response in the tissues and the PMN leukocytes will enter the milk in numbers direct proportion to the extent of the irritation.

Our test, therefore, is not a test for a specific type of udder irritation but rather a rapid procedure 5601' the detection of milks having abnormally high PMN leukocyte counts. It is a screening method for the detection of cows having irritated udders, and it provides a means for demonstrating in a visual manner the incidence and degree of udder irritations in a herd. Reacting udders should be tested further by bacteriologic methods to determine whether the irritation is of bacterial origin and for type of bacteria so that specific drugs and/or antibiotics may be selected by the veterinarian for treatment.

In the event that no pathogenic bacteria are found inglands producing positive milk, it may be interpreted that the irritation is the result of factors other than infection with the common mastitis bacteria, and proper steps other than therapy must be taken to overcome the inciting cause of the irritation.

We claim:

. 1. A method for testing for the presence of polymorph-onuclear leukocyte cells in a mammalian fresh milk comprising mixing substantially equal par-ts of the milk to be tested with a substantially pH neutral aqueous solution of an anionic surface active agent containing a trace of a pH indicator and noting the extent of thickening or development of viscosity in the resulting mixture.

2. A method for testing for the presence of poly morphonuclear leukocyte cells and other products of inflammation in milk comprising mixing substantially equal parts of the milk to be tested with an aqueous solution of an anionic surface active. agent containing a trace of pH indicator and noting the extent of thickening or development of viscosity in the resulting mixture.

3. r The method of claim l wherein said solution contains a trace of dye.

References Cited in the file of this patent UNITED STATES PATENTS Stalloch et al. Aug. is, 1950 

1. A METHOD FOR TESTING FOR THE PRESENCE OF POLYMORPHONUCLEAR LEUKOCYTE CELLS IN A MAMMALIAN FRESH MILK COMPRISING MIXING SUBSTANTIALLY EQUAL PARTS OF THE MILK TO BE TESTED WITH A SUBSTANTIALLY PH NEUTRAL AQUEOUS SOLUTION OF AN ANIONIC SURFACE ACTIVE AGENT CONTAINING A TRACE OF A PH INDICATOR AND NOTING THE EXTENT OF THICKENING OR DEVELOPMENT OF VISCOSITY IN THE RESULTING MIXTURE. 